Mechanisms of synergistic regulation of biliary inflammation and fibrosis Background: The risk of liver diseases due to alcohol and toxin abuse and hepatitis viruses in US Veterans is increasingly high and is one of the most common reasons for hospitalization and mortality. Chronic liver disease is characterized by damage to specific liver cellscollagen and matrix accumulation fibrosis and eventual scarring. Cholangiocytes are the target cells in cholangiopathies such as Primary Sclerosing Cholangitis (PSC) and Primary Biliary Cirrhosis (PBC), which are characterized by the proliferation/loss of cholangiocytes leading to fibrosis. Management of cholangiopathies represents one of the major challenges for Veterans. This proposal aims to increase our understanding of factors that regulate liver fibrosis and to improve patient care leading to more effective treatments for these disorders. Histamine (HA) is secreted by many cells including mast cells (MCs), but also is synthesized by cholangiocytes. We have shown that: (i) HA stimulates biliary proliferation/loss via interaction with specific HA receptors (HRs) and (ii) the enzyme responsible for HA synthesis, histidine decarboxylase (HDC) regulates biliary growth via interaction with and modulation of miR-125b and vascular endothelial growth factor (VEGF). The rationale for our proposal is built upon previously published data showing that HA levels increase in PBC and PSC patients and increased MC infiltration positively correlates with increased fibrosis. MCs, which interact with both cholangiocytes and hepatic stellate cells (HSCs) are the main source of HA, and may contribute to conditions like pruritus. Our overall goals of this project are to demonstrate that (i) HA (via specific HR interaction) is a fibrosis-promoting agent derived from several cellular sources including cholangiocytes and MCs; (ii) HA-mediated fibrosis is driven by the miR-125b/VEGF axis and (iii) cholangiocytes, MCs and HSCs synergistically regulate fibrosis. We will utilize both in vitro and in vivo models in our proposal. All of our established animal models are relevant to the proposal and support VA-centered research by mimicking human liver fibrosis that is seen in patients with both acute and chronic liver injury. Our preliminary data demonstrates that (i) HA promotes fibrosis and (ii) inhibition of mast cell-derived HA decreases fibrosis and hepatic stellate cell activation. We propose the novel hypothesis that synergistic regulation of HA contributes to hepatic inflammation and fibrosis progression via the miR-125b/HDC/VEGF axis. We propose the following aims: Specific Aim 1: To demonstrate that HA promotes hepatic inflammation and fibrosis. We will develop this aim by treating rodent models with HA and HA receptor agonists and evaluating the progression of fibrosis. Further, in models that mimic human liver fibrosis, we will measure the effects of blocking mast cell-derived HA and the potential therapies derived from blocking HA receptor activation on fibrosis. Specific Aim 2 - To determine the cellular mechanisms and potential therapies of HA-mediated liver fibrosis, in vivo and in vitro. To achieve this, we will use pharmacological and molecular approaches to block HDC and the specific HA receptors and evaluate the signaling pathway involved in hepatic fibrosis. In vitro, we will utilize established molecular biology techniques to evaluate the pathways that regulate HA-induced fibrosis. Specific Aim 3: To determine the cell-to-cell interaction of HA-mediated hepatic fibrosis between cholangiocytes, MCs and HSCs using various in vitro and in vivo models of hepatic fibrosis. Our working hypothesis is that following liver injury, bile ducts proliferate inducing MC recruitment, leading to an increase in HA release and HSC activation. We will evaluate these events in both relevant in vivo models of fibrosis and using in vitro cell cultures to study cell-to-cell interaction directly. The information gained upo the successful completion of these studies are expected to provide important insights into the role that histamine plays during hepatic fibrosis progression, which will ultimately help in the identification of important signaling pathways that can be targeted for the development of therapeutic interventions for fibrosis treatment.